The goal of our research is to define the functions of the coronavirus gene 1 proteins in viral replication. Mouse hepatitis virus (MHV) contains a 32 kb single-stranded, positive-sense RNA genome and synthesizes its polymerase gene products by translation and processing of an 803 kDa polyprotein from gene 1 of the input genome RNA. Many of the proteins processed from the gene 1 polyprotein have been identified, and the importance of the gene 1-encoded 3C-like proteinase in polyprotein processing has been established. However, many of the identified and predicted polyprotein cleavage products, including the putative RNA-dependent RNA-polymerase and helicase have not been experimentally confirmed. Furthermore, the importance of gene 1 proteins in formation and function of the MHV replication complex is not understood. This proposal describes a research program that will define the translation, processing, and localization of gene 1 proteins in virus infected cells. In addition, the gene 1 proteins in the viral replication complex will be identified. In Aim 1, the complete pattern of expression and processing of the gene 1 polyprotein will be determined in virus-infected cells, and the precise termini of the processed proteins will be identified. In Aim 2 immunofluorescence and laser confocal microscopy will be used to define the location of gene 1 proteins and their association with sites of MHV RNA synthesis and MHV structural proteins during MHV infection. In Aim 3 the viral protein, RNA, and membrane components of MHV replication complexes will be isolated from infected cells and the interactions of gene 1 proteins in the complexes will be defined. These experiments will precisely identify mechanisms by which coronaviruses regulate the availability and interactions of proteins essential for RNA transcription and replication and will determine how MHV proteins target intracellular sites of viral replication.